GB1589294A - Pharmaceutical compositions containing anthocyanidines - Google Patents

Description

PATENT SPECIFICATION ( 11) 1 589 294

( 21) Application No 37252/76 ( 22) Filed 8 Sep 1976 ( 19) Ch ( 23) Complete Specification Filed 7 Sep 1977

( 44) Complete Specification Published 13 May 1981

O ( 51) INT CL 3 A 61 K 31/35 It ( 52) Index at Acceptance A 5 B 170 180 380 381 382 38 Y 393 39 X - 482 48 Y 502 50 Y 550 55 Y 576 57 Y 586 58 Y 616 61 Y 650 65 Y FA H ( 72) Inventors: ANDREA LIETTI ATTILIO BOMATI ( 54) PHARMACEUTICAL COMPOSITIONS CONTAINING ANTHOCYANIDINES ( 71) We, INVERNI DELLA BEFFA S p A, an Italian Company of Via Ripamonti, 99, Milan, Italy, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:

This invention relates to pharmaceutical compositions and processes for their preparation 5 Anthocyanidines are a group of known polyphenolic substances These products, as well as being preparable by total chemical synthesis, may be obtained by hydrolysis of their glycosides which are widely distributed in nature These glycosides, and particularly the glucosides, are known as anthocyanins and are present in the fruits of the bilberry, vine, elder, currant, bramble and raspberry 10 We have now found that anthocyanidines are endowed with remarkable cicatrising and epithelium-regenerating properties which render them particularly useful in the treatment of cutaneous wounds, torpid sores, and external and internal ulcers Also anthocyanidines have been found to possess distinct anti-inflammatory, vaso-protective, hypolipaemic, hypocholesterolaemic and hypoglycaemic activities 15 Anthocyanidines frequently have extremely low toxicities which renders them particularly useful for prolonged treatments.

Clinically, the substances may be administered singly or in the form of mixtures with one another, in the pure state or in the form of crude or partially purified extracts, for example extracts of the crude product obtained by hydrolysis of naturallyoccurring mixtures of 20 anthocyanins Preferably at least a portion of extracted materials other than the anthocyanidines are eliminated from the extracts.

Thus according to the present invention there are provided pharmaceutical compositions comprising in admixture with a pharmaceutically acceptable diluent or carrier, an anthocyanidine, or a pharmaceutically acceptable salt thereof, with the proviso that when the 25 sole diluent or carrier is a liquid solvent, the composition is sterile and pyrogen-free.

Preferably, since they may be obtained readily by hydrolysis of glycosides contained in common fruit and also on account of their pronounced pharmaceutical activity, the active constituent or constituents contained in the pharmaceutical compositions of the invention are cyanidine, peonidine, delphinidine, petunidine, pelargonidine or malvidine 30 These anthocyanidines, which may be in the form of salts with any pharmaceutically acceptable anion (e g chloride, sulphate, phosphate, acetate or hydroxyl), may be represented by the following formula:

R 1 X 35 HO R 2 OH 40 OH wherein: R' = R 2 = -OH and R 3 = H(cyanidine) R= -O Me, R 2 = -OH and R = H(peonidine) R 1 = R 2 = R 3 = OH (delphinidine) 45 2 1,589,294 2 R' = O Me and R 2 = R 3 = OH (petunidine) W= R 3 = -O Meand R 2 = -OH (malvidine) R 2 = -OH and R= R 3 = H (pelargonidine) and X is the pharmaceuticallly acceptable anion.

The particular galenic form of the compositions of the invention depends on the intended 5 route of administration and the condition to be treated and such forms may be amorphous or in the form of shaped dosage units Examples include sterile liquids suitable for parenteral administration, forms suitable for oral administration (e g tablets, capsules, solutions or suspensions); forms suitable for insertion into a body cavity (e g anal or vaginal suppositories); forms suitable for topical administration (e g ointments, creams, gels and aque 10 ous solutions or suspensions) and dentifrices.

In formulating compositions according to the invention, a wide range of excipients may be used, the nature of which will depend, of course, on the intended mode of application of the composition Examples include preservatives and buffering, thickening, suspending, stabilising, wetting, emulsifying, colouring and flavoring agents and in particular carboxy vinyl 15 polymers, propylene glycol, ethyl alcohol, water, cetyl alcohol, saturated vegetable triglycerides, fatty acid esters or propylene glycol, triethanolamine, glycerol, starch, sorbitol, bentonite, carboxymethyl cellulose, laurylsulphate, dicalcium phosphate, powdered silica and lecithin.

Frequently, more than one diluent or carrier is advantageously used 20 The compositions of the invention have been found to be particularly useful in the treatment of wounds, gastric and duodenal ulcers, inflammatory conditions of the mouth and throat, pathogenic conditions of the vascular system and disorders caused by impaired lipidic and glycidic metabolism.

This invention also includes a process for producing the compositions defined above which 25 comprises extracting an anthocyanin from plant tissue, hydrolysing the anthocyanin to form an anthocyanidine, purifying the anthocyanidine and admixing the purified anthocyanidine in free form or in the form of a pharmaceutically acceptable salt with a pharmaceutically acceptable excipient.

The invention also provides a method of inducing a cicatrising, epithelium regenerating, 30 anti-inflammatory, vaso-protective, hypolipaemic, hypocholesterolaemic or hypoglycaemic response in a non-human subject, which comprises administering to the subject a dose of an anthocyanidine in an amount sufficient to produce the required response.

The compositions according to the invention preferably contain at least 0 2 % and most preferably at least 0 5 % by weight of anthocyanidines More concentrated compositions are 35 preferred for internal (i e enteral or parenteral) administration, particularly those containing at least 1 % and more particularly at least 5 % by weight of anthocyanidines The compositions may be administered at a daily dosage rate of from 1 to 100 mg/kg, preferably 5 to 50 mg/kg of anthocyanidines.

The following experimental data illustrates the pharmacological properties of 40 anthocyanidines:

I Anti-ulcer activity Shay's ulcer in the rat In Shay's ulcer in the rat, the oral administration of bilberry anthocyanidines at doses of 25 to 50 mg/kg five times, at 48,33,22 and 9 hours prior to the ligature of the pylorus and 1 hour after the operation, was found to diminish the ulcer index observed with the controls, by 28 45 and 39 per cent respectively (See Table 1).

Table 1 Anti-ulcer activity Shay's gastric ulcer in the rat TREATMENT Number of ulcers ofeach class ( 2) Variation of Number Dose Number Ulcer Ulcer Index nonmg/kg of Index ulcerated animals stomachs ( 1) I II III IV V ( 3) Controls 19 295 ( 295) 44 ( 220) 15 ( 150)2 ( 40)17 ( 340) 55 (water) 18 100 ( 100) 20 ( 100) 13 ( 130) 9 ( 180) 10 ( 200) 39 5 -28 11 Bilberrry anthocyanidines 18 271 ( 271) 21 ( 105) 7 ( 70) 3 ( 60) 5 ( 100) 33 6 -39 5 ( 1) Doses administered orally 5 times, 48,33,22 and 9 hours prior to ligature of the pylorus and 1 hour after ( 2) In parentheses, the product of the number of the ulcers and the value of the individual classes according to the evaluation criterion of Keyriliinen T O and Passonen M K.

(Acta Pharmacol et Toxicol 13,22,1957) ( 3) Percentage reduction compared with controls CJ 1 00 ,.O to) ,D 4 1,589,294 4 II Activity upon lipidic metabolism 1) Hyperlipaemia induced by olive oil Hyperlipaemia was induced by orally administering olive oil to male Sprague-Dawley rats of mean weight of 165 g which had been fasted for 16 hours The administration of olive oil was effected 3 hours prior to sacrifice, at a dose of 2 ml/gk orally 5 Bilberry anthocyanidines and cyanidine obtained from elder fruit were administered intraperitoneally 1 hour prior to the administration of the olive oil in equal strength doses dissolved in 0 5 ml/kg of physiological solution Table 2 shows that the bilberry anthocyanidines significantly diminish the free fatty acids and the triglycerides respectively by 36 60 and 86 % in comparison with the controls, and that the cyanidine obtained from 10 elder diminishes the free fatty acids and the triglycerides respectively by 32 70 and 67 90 %, again in comparison with the controls.

2) Hyperlipaemia induced by Triton WR 1339 Hyperlipaemia was induced by intravenous administration of 225 mg/kg, 0 5 ml/kg of Triton WR 1339 dissolved in physiological solution, in male SpragueDawley rats of mean 15 weight 210 g which had been fasted for 24 hours, 8 hours prior to sacrifice ("Triton" is a Registered Trade Mark) The substances under test were injected intraperitoneally twice: the first administration simultaneously with the Triton and the second 4 hours later, in equalstrength doses dissolved in 0 5 ml/kg of physiological solution 20 From Table 3 it can be seen that bilberry anthocyanidines significiantly diminish the plasma triglycerides and cholesterol respectively to 29 56 and 16 03 %in comparison with the controls and that elder cyanidine diminishes these lipids by 23 65 and 16 67 % respectively, again in comparison with the controls.

Table 2 Effect upon hyperlipaemia induced by olive oil 25 TREATMENT Dose Number NEFA Triglycerides mg/kg of l Eq/l mg/100 ml animals 30 A Controls 16 1013 22 + 44 30 384 52-+ 42 34 B 58 %antho 43 1 16 642 33-+ 27 44 53 73 + 6 24 cyanidines (-36 60) (-86 00) from bilberry 35 C 32 %cyanidine 72 12 16 681 73-+ 25 21 123 62 + 36 68 from elder (-32 70) (-67 90) Non-esterified fatty acids 40 0 Significantly different (p< O 001) from the mean obtained from group A according to Student's "t" test Note: In parentheses, the percentage difference from the controls.

Table 3 Effect upon hyperlipaemia induced by Triton WR 1339 45 TREATMENT Dose Number Triglycerides Total cholesterol mg/kg of mg/100 ml mg/100 ml 50 animals A Controls 8 621 56 ±36 87 189 93 + 11 76 B 58 %Antho 21 5 x 2 8 437 85 + 26 0100 159 50 + 3 20 cyanidines (-29 56) (-16 03) from bilberry 55 C 32 %cyanidine 36 6 x 2 8 474 59 + 38 06 158 28 + 6 58 from elder (-23 65) (-16 67) Significantly different (p < 0 05) from the mean obtained with the controls according to 60 Student's "t" test 00 Significantly different (p < 0 01) from the mean obtained with the controls according to Student's "t" test Note: In parentheses the percentage difference from the controls 65 1,589,294 5.

III Activity upon capillarypermeability The action upon capillary permeability was studied on Sprague-Dawley rats of mean weight of 220 g which had been fasted 18 hours prior to the experiment according to the method of Ankier and West; Brit J Pharmacol 33, 304, 1968 From Table 4 it can be seen that bilberry anthocyanidines administered experimentally by intraperitoneal route at doses 5 of 9,18 and 36 mg/kg give a significant diminution of the capillary permeability respectively by 12; 25 2 and 55 4 %in comparison with the controls.

Table 5 shows the experimental data obtained by oral treatment with bilberry anthocyanidines The produce was administered experimentally in two doses and specifically at 36 and 72 mg/kg, and gave a significant inhibition of the capillary permeability of 24 6 and 10 44.4 %in comparison with controls.

Table 4 Capillary permeability induced by bradykinin in the rat TREATMENT Evans Blue Inhibition mg/kg /g percent 15 Controls O 5 ml/hg 16 87 O 20 0.9 %Na Cl 70 %antho 9 12 20 cyanidines frombilberry 18 12 63 + O 100 25 2 36 7 54 0 30 55 4 Note: Bradykinin 2 gg/0 1 ml injected intradermally at 3 points into the depilated abdomi 25 nal zone of each rat Treatment with the substances under examination 30 minutes prior to the bradykinin Sacrifice 30 minutes after the bradykinin 30 o Significantly different (p < 0 01) from the mean obtained with the controls according to Student's "t" test Table 5 Capillary permeability induced by bradykinin in the rat 35 TREATMENT Evans Blue Inhibition mg/kg,ug per cent Controls(H 20) 1 ml/hg 15 50 O 54 40 % antho 36 11 70 + 0 51 24 6 cyanidines frombilberry 72 8 60 0 58 44 4 45 Note: Bradykinin 2,g/0 1 ml injected intradermally at 3 points of the depilated abdominal zone of each rat Treatment with the substances under examination 60 minutes prior to the bradykinin Sacrifice 30 minutes after the bradykinin 50 o Significantly different (p < 0 01) from the mean obtained with the controls according to Student's "t" test Activity upon capillary resistance The capillary resistance was studied in rats subjected to deficiency diet according to the 55 method of Charlier R et al; Arch intern Physiol Biochem, 71,1, 1963.

Table 6 shows that the bilberry anthocyanidines used experimentally in two doses by oral route increased the capillary resistance with time and in significant manner.

1,589,294 6,8,24 ' Table 6 Activity upon the capillary resistance of rats subjected to dietary deficiency TREATMENT Time from treatment 5mg/kg/os hours 6 0 2 4 6 %anthocyanidines 36 15 5 + 0 32 17 0 0 32 17 2 + 0 14 17 2 + 0 14 from bilberry 72 15 5 + 0 32 17 3 0 41 17 6 + 0 41 17 6 + 0 41 10 Significantly different from the time O (p< 0 05) according to Student's "t" test for non-independent samples 15 Table 7 Effect upon hyperlipaemia induced by olive oil TREATMENT Dose Number NEFA Triglycerides 20 of i Eq/1 Animals A Controls(Na C 10 9 %) lml/hg 8 1056 5 + 29 3 312 8 + 33 6 B Pelargonidinechloride 100 mg/kg 8 671 5 27 4 (-36 4) 140 4 + 11 6 (-55 1) C Peonidine 100 mg/kg 8 950 5 + 22 9 (-10 0) 176 0 + 21 3 (-43 8) 25 Non-esterified fatty acids.

O Significantly different (p < 0 05) from the mean obtained from group A to Student's "t" 30 test.

Note: In parentheses, the percentage difference from the controls.

The anthocyanidines suitable for the preparation of the composition according to the invention may be obtained by hydrolysis of anthocyanins contained in plant extracts or by 35 synthetic methods Generally, plants containing anthocyanins are extracted with alcoholic solvents (particularly low alkanols containing 1 to 4 carbon atoms) containing small quantities of mineral acids which stablise the pyran form of the anthocyanic nucleus.

The warming of the extract with acid causes hydrolysis of the glycoside residue of the anthocyanins thus forming anthocyanidines which may be incorporated into the composi 40 tions of the invention either as mixtures of after having being isolated as pure products using column chromatography.

Examples 1 and 2 below describe the preparation of anthocyanidine from elder fruits and of mixtures of five anthocyanidines (malvidine, delphinidine, cyanidine, peonidine and petunidine) from a methanol extract of bilberry fruits 45 By synthetic methods flavones (L Bauer, Chem and Ind 433, 1954 and H G C King, J.

Chem Soc 901, 1957), catechins (J Savollary, Compt Rend 217, 86, 1943 and H Apple J.

Chem Soc 426, 1935) and oligomers of catechins (T A Geissman and H F K Dittmar, Phytochemistry 4,359, 1965) may be transformed into anthocyanidines.

Besides, it is possible to obtain anthocyanidines by condensing 2,4,6-tri 50 hydroxybenzaldehyde 2-0-benzoate and an omega-acetoxy-acetophenone suitably substituted in the aromatic nucleus according to the methods of R Robinson (A Robertson and R.

Robertson, J Chem Soc 1526, 1928 A Robertson, R Robinson and J Sugiura, J Chem.

Soc 1533,1928 S Murakami and R Robinson, J Chem Soc 1537,1928 W Bradley and R.

Robertson J Chem Soc 1541, 1928) Example 3 describes the synthesis of pelargonidine 55 chloride.

Example 1 -Preparation of anthocyanidines from elder anthocyanins (A) Extraction from elder 13.5 kg of fresh ripe elder fruits were extracted at room temperature with anhydrous methanol containing 1 % of hydrochloric acid The extracts were concentrated in vacuo to 60 small volume and an aqueous solution of 30 % of neutral lead acetate added with agitation.

An abundant precipitate was obtained which was filtered and washed with water 200 g of crude lead salts were obtained which were then suspended with agitation in 600 ml of anhydrous methanol containing hydrochloric acid, agitated at room temperature and the insoluble material elminated by filtration 65 1,589,294 1,589,294 The methanolic solution was then concentrated in vacuo at low temperature to small volume and subsequently poured with agitation in to ether The precipitate was filtered and dried in vacuo at room temperature 30 g of glucosides were obtained, equivalent to 15 % cyanidine.

(B) Formation ofcrude cyanidine 5 g of glucosides equivalent to 15 % cyanidine were dissolved in a mixture constituted by methanol and concentrated hydrochloric acid in the ratio 8: 2 Heating was effected under reflux for 3 hours The solution was then diluted wih water and concentrated in vacuo until complete elimination of the methanol A precipitate was obtained which was filtered and washed with water After drying, 6 g of crude hydrolysate was obtained containing 20 % 10 cyanidine.

(C) Purification ofthe cyanidine The crude 20 % cyanidine was purified by chromatography on Sephadex LH 20, eluting with 95 % ethanol containing 1 % of concentrated hydrochloric acid ("Sephadex" is a Registered Trade Mark) 15 Although anthocyanidines for incorporation in pharmaceutical compositions according to the invention are most conveniently prepared by hydrolysis of fruit anthocyanins (anthocyanosides), for example as described above, they may also be obtained synthetically by reduction of quercetin and its derivatives or rutin (L Bauer, Chem and Ind 1954,433-4; H G C King, J Chem Soc 1957, 3901-3) Other methods reported in literature enable 20 cyanidine to be obtained from epicatechin pentaacetate (A K Ganguli et altri Proc Indian Aca Sci, 46 A, 25-8, 1957) and from catechin and its derivatives (J Lavollary, Compt Rend.

217,86-8,1943; H Apple J Chem Soc 1935,426-9).

Moreover cyanidine can be obtained by acid hydrolysis of oligomers (polymers of low molecular weight, generally dimers) of catechins (T A Geissman and H F K Dittmar 25 Phytochemistry, 1965, vol 4 pp 359-368).

Example 2 -Preparation of anthocyanidines from bilberry Using the procedure used in Example 1, an extract was obtained rich in anthocyanosides equivalent to not less than 25 % in total anthocyanidines (malvidine, delphinidine, cyanidine, peonidine and petunidine) 30 Subsequently the extract was hydrolysed and the anthocyanidines are purified by the following procedure:

The anthocyanosides were dissolved in a mixture of methanol and concentrated hydrochloric acid in the ratio 8: 2, and hydrolysed by means of boiling under reflux for 3 hours The precipitate formed was cooled and filtered The liquid was then concentrated in vacuo to 35 eliminate all the methanol, and four extractions with isoamyl alcohol were effected on the concentrate The reunited isoamyl extracts were concentrated in vacuo and precipitated with agitation in ethyl ether After filtration and drying, anthocyanidines at 50 60 per cent were obtained.

Example 3 Synthesis of Pelargonidine Chloride 40 24 g of 2,4,6-trihydroxybenzaldehyde 2-0-benzoate were dissolved by warming in 500 ml of ethyl acetate and 20 g of p-hydroxyacetoxyacetophenone added Gaseous hydrochloric acid was introduced to saturation and the mixture kept at room temperature overnight The obtained solid was then dissolved in 3 litres of methanol, 150 ml of conc aqueous HCI added and the mixture boiled under reflux for 6 hours The product was then evaporated under 45 vacuum to 250 ml and allowed to crystallise at 4 C Yield 14 g of pelargonidine chloride.

Pharmaceutical compositions according to the invention was prepared in accordance with the following formulations:

50Injectable solution forfreeze-drying 5 Bilberry anthocyanidines ( 50 %by weight) 25 mg Excipients (mannite, sodium chloride, sodium ethylene diamino tetraacetate, thiourea) q sto 125 mg Solvent: double-distilled water (pyrogen-free) 3 m 55 Capsules Grade anthocyanidines ( 25 %by weight) 100 mg Excipients (mannite, citric acid, sodium chloride, thiourea, sodium ethylene diamino tetraacetate, lactose, methyl cellulose, magnesium stearate) q sto 200 mg 60 (The components were mixed together and then filled into capsules.

1,589,294 Capsules Elder anthocyanidines (containing 20 %cyanidine) 125 mg Excipients (mannite, citric acid, sodium chloride, thiourea, sodium ethylene diamino tetraacetate, lactose, methyl cellulose, magnesium stearate) q s to 250 mg (The components were mixed together and then filled into capsules) Injectable solution for freeze-drying 10Cyanidine 15 mg 10 Excipients (mannite, sodium chloride, sodium ethylene diamino tetraacetate, thiourea) q sto 125 mg Solvent: double-distilled water (pyrogen-free) 3 ml 15 ' Tablets

Grape anthocyanidines ( 60 %by weight) 35 mg Excipients (maize starch, lactose, citirc acid, magnesium stearate, thiourea, sugar, talc, gum arabic, magnesium carbonate) q s to 200 mg 20 Ointment Bilberry anthocyanidines ( 50 % by weight) 0 5 g Excipients (cetyl alcohol, saturated vegetable triglycerides, esters of polyethylene glycol 2000 with fatty 25 acids C 12-CM 4, Tween 80, para-oxy benzoates, sorbitol, carboxy vinyl polymer, sodium sulphite, triethanolamine, lecithin, purified water, lactic acid) q sto 100 g Ointment Elder anthocyanidines (containing 20 %cyanidine) 1 g 30 Excipients (cetyl alcohol, saturated vegetable triglycerides, esters of polyethylene glycol 2000 with fatty acids C 12-C 14, Tween 80, para-oxybenzoates, sorbitol, carboxyvinyl polymer, sodium sulphite, triethanolamine, 35 lecithin, purified water, lactic acid) q sto 100 g Dentrifice gel Bilberry anthocyanidines ( 35 %by weight) 0 5 g Excipients (carboxyvinyl polymer, sorbitol, propylene glycol, sodium sulphite, ethyl alcohol, para-oxy ben 40 zoates, sodium lauryl sulphate) q sto 100 g Dentifrice paste Grapeanthocyanidines( 60 %byweight) 0 5 g Excipients (citric acid, sodium bisulphite, sorbitol, 45 ammonium glycyrrhizinate, maize starch, glycerine, para-oxy benzoates, titanium dioxide, calcium phosphate, sodium lauryl sulphate, flavourings, purified water) q sto 100 g (Conventional techniques were used to prepare the tablet, ointment and dentifrice formulations from the ingredients specified) 50

Claims (1)

1. WHAT WE CLAIM IS:-

   1 A pharmaceutical composition comprising a pharmaceutically acceptable excipient and as active ingredient an anthocyanidine or a pharmaceutically acceptable salt thereof, with the proviso that where the sole excipient is a liquid solvent, the composition is sterile and 55 pyrogen-free.

   2 A composition according to Claim 1 containing a preservative or a buffering, thickening, suspending, stablizing, wetting, emulsifying, colouring or flavouring agent.

   3 A composition according to Claim 1 or Claim 2 comprising excipient selected from carboxy vinyl polymers, propylene glycol, ethyl alcohol, water, cetyl alcohol, saturated 60 vegetable triglycerides, fatty acid esters, propylene glycol, triethanolamine, glycerol, starch, sorbitol, bentonite, carboxymethyl cellulose, laurylsulphate, dicalcium phosphate, powdered silica and lecithin.

   4 A composition according to any preceding claim comprising at least two excipients.

   5 A composition according to any preceding claim in which the anthocyanidine is 65 9 1,589,294 9 selected from cyanidine, peonidine, delphinidine, petunidine, malvidine and pelargonidine.

   6 A composition according to any preceding claim in a form suitable for parenteral administration.

   7 A composition according to any one of Claims 1 to 5 in a form suitable for oral administration 5 8 A composition according to any one of Claims 1 to 5 in the form of a suppository.

   9 A composition according to any one of Claims 1 to 5 in the form suitable for topical administration.

   A composition according to any one of Claims 1 to 5 in the form of a dentifrice.

   11 A composition according to any one of Claims 1 to 5 in the form of a dosage unit 10 12 A composition according to any one of Claims to 11 containing at least O 2 wt %of anthocyanidines.

   13 A composition according to Claim 12 containing from 1 to 50 wt % of anthocyanidines.

   14 A composition according to Claim 13 containing at least 5 wt % ofanthocyanidines 15 A process for producing a pharmaceutical composition as claimed in any preceding claim which comprises extracting an anthocyanin from plant tissue, hydrolysing the anthocyanin to form an anthocyanidine, purifying the anthocyanidine and admixing the purified anthocyanidine in free form or in the form of a pharmaceutically acceptable salt with a pharmaceutically acceptable excipient 20 16 A method of producing a cicatrising, epithelium regenerating, antiinflammatory, vaso-protective, hypolipaemic, hypocholesterolaemic or hypoglycaemic response in a nonhuman subject, which comprises administering to the subject dose of an anthocyanidine in an amount sufficient to produce the required response.

   17 A method according to Claim 16 in which the anthocyanidine is selected from 25 cyanidine, peonidine, delphinidine, petunidine, malvidine and pelargonidine.

   18 A method according to Claim 16 in which the anthocyanidine is administered in the form of a composition as claimed in any one of Claims 1 to 14.

   19 A method according to Claim 16 in which the anthocyanidine is administered in the form of a composition produced by a process as claimed in Claim 15 30 A method according to any one of Claims 16 to 19 in which the anthocyanidine is administered to an animal afflicted with wounds, gastric and duodenal ulcers, inflammatory conditions of the mouth and throat, pathogenic conditions of the vascular system or disorders caused by impairment of lipidic or glycidic metabolism.

   21 A method according to any one of Claims 16 to 20 in which said effective dose is from 35 1 to 100 mg/kg per day of anthocyanidines.

   22 A method according to claim 21 in which said effective daily dose is from 5 to 50 mg/kg per day of anthocyanidines.

   MATHYS & SQUIRE, Chartered Patent Agents, 40 Fleet Street, London E C 4 Agents for the Applicants Printed for Her Majesty's Stationery Office, by Croydon Printing Company Limited, Croydon, Surrey, 1981.

   Published by The Patent Office, 25 Southampton Buildings, London, WC 2 A l AY, from which copies may be obtained.